Most flaviviruses are transmitted horizontally between vertebrate hosts and hematophagous arthropods; thus, these viruses are exposed to fitness constraints imposed by disparate biological systems that preclude specific adaptation to either host. In contrast, other recently described flaviviruses, such as Culex flavivirus (CxFV), have no known vertebrate host and are considered to be insect-specific. Others, such as Modoc virus (MODV), are vertebrate-specific. Viruses in the latter two groups have the potential for host-specific adaptation independent of the need to maintain a compromised fitness level in each host in order to maximize their overall fitness. The evolutionary processes and underlying genetic basis for the differential host ranges and transmission cycles of these viruses have yet to be addressed; however, the presence of both dual-host (vertebrate-mosquito) and single-host (mosquito-specific and vertebrate-specific) flaviviruses makes this an optimal system for addressing some of these evolutionary constraints and the resulting genetic determinants that modulate host specificity. Thus, the overall goal of this application is to identify the genetic elements that have conditioned the differential host-ranges, transmissibility's and tropisms of dual-host and single-host Flaviviruses. Three specific aims have been designed to achieve this goal. In specific aim 1, the genetic elements that modulate the differential host ranges of dual-host and insect-specific flaviviruses will be identified by assessing the in vitro and in vivo vertebrate and mosquito infection phenotypes of chimeric viruses created between WNV and CxFV. In specific aim 2, the genetic determinants that modulate flavivirus host range will be further defined by characterizing the in vitro and in vivo replication phenotypes of viral chimeras created between a dual-host and vertebrate- specific flavivirus (WNV and MODV, respectively). In specific aim 3, two recently discovered ribosomal frameshift proteins - one uniquely encoded by all known insect-specific flaviviruses and the other by WNV and all other Japanese encephalitis serogroup viruses - will be investigated in order to determine their significance in flavivirus host adaptation.